Cytosolic phosphodiesterase activities of Jurkat, a human T-lymphocyte cell line, were resolved by high performance liquid chromatography into two major peaks of cAMP-specific enzyme. One was the Rolipram-sensitive isozyme. The other showed a low Km value (0.45 ± 0.04 μM) for cAMP and different properties from reported isozymes in that it was hardly affected by well-known phosphodiesterase inhibitors or activators. This enzyme, 'JK-21', was found in other human T-cell lines, MOLT-4, HPB-ALL and HUT-78, whereas no fraction containing JK21 was detected in human B-lymphocyte cell lines, Jijoye, JY and Namalwa and in human promyelocytic cell line, HL-60. The results indicate that JK-21 is a new phosphodiesterase isozyme localized to T-cells. © 1993 Academic Press, Inc.